WO1999016908A2

WO1999016908A2 - Display technique for identifying line-1 insertion site polymorphisms

Info

Publication number: WO1999016908A2
Application number: PCT/US1998/020620
Authority: WO
Inventors: Gary D. Swergold; Fang-Miin Sheen
Original assignee: THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES 6011 Executive Boulevard, Suite 325
Priority date: 1997-09-29
Filing date: 1998-09-29
Publication date: 1999-04-08
Also published as: WO1999016908A3; AU9596098A

Abstract

This invention relates to a novel display technique called LINE-1 Display which is designed to easily identify differences in the L1Hs insertion sites between different individuals. LINE-1 Display is capable of detecting two types of polymorphisms: differences in insertion site locations, and differences in the lengths of L1Hs polyA tails. Insertion site polymorphisms are particularly useful for human molecular anthropology while length polymorphisms are particularly useful for genetic mapping studies.

Description

DISPLAY TECHNIQUE FOR IDENTIFYING LINE-1 INSERTION SITE POLYMORPHISMS

FIELD OF THE INVENTION

The present invention pertains, in general, to methods of human identification by DNA analysis. In particular, the present invention pertains to a novel PCR-based method called

LINE-1 Display which reveals frequent insertion site polymorphisms in the human population.

BACKGROUND OF THE INVENTION

All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. A transposable element or transposon is a DNA sequence able to insert itself at a new location in the genome without having any sequence relationship with the target locus. Transposons are an important force for change in both prokaryotic and eukaryotic genomes. Eukaryotic transposable elements are divided into two general groups. One group of elements is comparable to bacterial transposons . The other group of elements is similar to retroviruses in their general organization and by their use of RNA intermediates for transposition. Because of their relationship to retroviruses, these elements are called retrotransposons or retroposons. Retrotransposons differ from retroviruses in that the retrotransposons do not pass through an independent infectious form. Both transposon classes, the bacterial-like transposons and the retrotransposons, generate short direct repeats of target DNA when an insertion occurs (see reference 34 for a general discussion of retroviruses and retroposons) .

A large portion of the moderately repetitive DNA of mammalian genomes consist of retrotransposons. The major types of retrotransposons consist of short interspersed sequences or elements (SINES) and long interspersed sequences or elements (LINES) . The most abundant family of SINES found in mammals consists of the Alu repeats, which are present at about a million copies per genome. These SINES are collectively called the "Alu family" because they usually contain the recognition sequence AGCT for the restriction enzyme Alul. Methods have been developed for generating polymorphic markers based on the amplification of microsatellites at the 3 ' end of Alu sequences (36) .

Mammalian genomes also contain 20,000 to 100,000 copies of LINES called LINE-1 or LI. All mammalian species have both older elements and younger, actively transposing LINES. For most species the identity of the actively mobile groups is not known. A typical LI is approximately 6,000 to 7,000 kb long, terminates in an A-rich tract, and may have open reading frames. Human LI has an internal RNA polymerase promoter and two open reading frames (31-32) . The first reading frame encodes a protein (p40) of ca . 40 kd with no known function (32) . Antibodies having binding affinity to the p40 protein have been produced and isolated (35) . Human LINE-1 sequences (LlHs) make up about 5% of the human genome and most are defective, primarily due to truncation and internal rearrangements (33) . The average human haploid genome harbors an estimated

100,000 truncated and 15-30 active LINE-1 (LlHs) retrotransposons (1-3) . All but one of the known LlHs insertion site polymorphisms and new transpositions belong to a subset of LlHs called Ta which is identifiable by sequence signatures in the 3' UTR. We used quantitative Southern blotting to estimate that 2250 subset Ta 3' UTRs exist in the human genome.

LINE-1 elements are sequences of DNA that are commonly known as jumping genes. They have the ability to create new copies of themselves and to insert the new copies into places in the DNA where they were not previously located. Individual persons differ in the location of many of the LINE-1 elements in their DNA. In addition, alleles differing in the length of LlHs poly-A tails exist. These differences contribute to the genetic differences between people. Genetic variation is an important determinant of the differences that make all people individuals (our phenotypes) . It contributes to our differential responses to environmental exposures, and drug sensitivities. It also allows individuals to be identified by their DNA. Here we report a novel technology called LINE-1 (LI) display designed to identify loci that differ by the presence or absence of an insertion (i.e., dimorphic sites). To test LI display we investigated the variability of LlHs insertion sites in the DNAs of 6 male individuals from diverse ethnic backgrounds. Of the previously reported 5 LlHs insertion site dimorphisms and 7 de novo transpositions, all but one belong to the Ta subset of elements (3-7) . In the present study, greater than 20% of the Ta sites tested and few of the non-TA sites were dimorphic. We isolated 6 dimorphic TA insertion sites and confirmed their status by Southern blotting, PCR with flanking primers, or both. Quantitative Southern blotting suggested that about 4,500 Ta 3' UTRs, at least 900 of which are dimorphic, exist in an average diploid human genome. LI display represents a powerful new method for isolating dimorphic LlHs insertion sites which will be a valuable tool for the investigation of genomic structure and evolution (8) . Our invention is designed to compare the DNA from different individuals, identify differences in the location of LINE-1 elements in their DNA and clone the insertion sites. This is the first report of a method designed to identify insertion site differences directly. By discovering these differences we will be able to develop better means for identifying individuals and determining their ethnic backgrounds from their DNA. This method can also be used to identify the actively mobile LINES in a species for which this information is not known. LINE-1 insertion polymorphisms will be important in understanding how genetic variation contributes to phenotype . The methods of this invention will help identify people at increased risk for disease and help to target the use of drugs more specifically to the appropriate patients.

SUMMARY OF THE INVENTION

This invention comprises methods of human identification by analysis of DNA. More specifically, the present invention provides a novel display technique called LINE-1 Display which is designed to easily identify differences in the LlHs insertion sites between different individuals. LINE-1 Display is capable of detecting two types of polymorphisms: differences in insertion site locations, and differences in the lengths of LlHs A-rich tails. LINE-1 Display represents a powerful new tool for identifying both rare and common DNA polymorphisms.

This invention provides display techniques for identifying LINE-1 insertion site polymorphisms used for the forensic identification of individuals, body fluids, body parts, etc. In addition, this invention provides methods for the identification of donated cells or organs as to the individual of donation. Even further, this invention provides methods used to identify the ethnicity of a person on the basis of DNA analysis. Prior art methods of using DNA markers are not easily capable of accomplishing the results which can be obtained using the present invention.

This invention provides display techniques for identifying LINE-1 insertion site polymorphisms used in genealogy. More specifically, this invention provides methods of using a sample of cells or blood or body fluids to identify an individual's ethnic background (s) . This invention further provides methods of determining the background of individuals who are interested in their past histories or for whom the information is lost.

The present invention also provides display techniques for identifying LINE-1 insertion site polymorphisms used for determining the relationships between genetic variation and disease susceptibilities. The prior art teaches that researchers are increasingly investigating the contribution of genes to the propensity for acquiring specific diseases. The methods of the present invention permit the identification of the direct contributions made by transposable element polymorphisms to disease susceptibilities and any associations between LINE insertions and disease susceptibility.

The present invention also provides display techniques for identifying LINE-1 insertion site polymorphisms used for identifying the relationships between genetic variation and drug responses. The methods of the present invention permit the discovery of differential responses, both intended and adverse, to the administration of drugs by transposable element polymorphisms . The present invention also provides display techniques for identifying LINE-1 insertion site polymorphisms used for the identification of stored body parts. In addition, the methods of the present invention will allow the positive identification of body parts, including organs and cells, which have been stored for prolonged periods. Thus, the methods of the present invention will permit the positive identification of body parts subjected to long-term storage prior to their administration to recipient patients. The present invention also provides display techniques for identifying LINE-1 insertion site polymorphisms used for cancer- related research, diagnosis, and treatment. For example, LINE-1 transposition may be involved in the development of certain types of cancer, especially breast and testicular. The methods of the present invention allow this hypothesis to be investigated. If there is a relationship then our mapping method would be useful as a clinical test for margins of resection, for early diagnosis, determining the risk of disease, etc. The monitoring of LINE-1 transposition would be a useful marker in developing breast cancer prophylaxis.

The present invention also provides display techniques for identifying LINE-1 insertion site polymorphisms used for the study, detection, diagnosis, and treatment of certain developmental abnormalities. For example, LINE-1 insertion may be involved in the development of certain developmental abnormalities. Thus, LINE-1 insertion may contribute to the unexplained loss of fetuses and to the development of aneuploids by non-disjunction. If this is true the methods of the present invention would be useful clinically for explaining fetal loss and for developing methods to combat fetal loss (i.e. drug development) .

Our analysis reveals that LlHs insertion sites are highly polymorphic in the human population. Thus, LINE-1 Display represents a powerful new tool for identifying both rare and common DNA polymorphisms. LlHs insertion site polymorphisms identified by the methods of the present invention will be valuable tools for the field of human molecular anthropology. In addition, LlHs insertion site and tail length polymorphisms identified by the methods of the present invention will be useful for genetic mapping studies.

One skilled in the art can easily make any necessary adjustments in accordance with the necessities of the particular situation. For example, the methods of the present invention can be easily modified to enable the identification of specific non-human mammals. As discussed above, all mammalian genomes contain LINES. Non-human mammal identification by DNA analysis using LINE-1 Display has application in a number of areas, including the tracking of tainted or diseased meat supplies. LINE-1 Display will also be useful for evolutionary studies of non-human mammals. Further objects and advantages of the present invention will be clear from the description that follows .

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. LI display protocol. A truncated of full-length LIHs-Ta (rectangle) is depicted surrounded by flanking DNA (solid lines) . The relative locations of the ACA, 6015G (see Materials and Methods) , and Ace I sites are indicated. The dashed lines represent the products of 2 rounds of PCR amplifications. In each round, multiple separate reactions were performed. In the first round, each reaction contained a Ta- specific primer (ACA) and a single arbitrary 10 bp primer. In the second round, portions of each of the first round reactions were reamplified using a nested primer (NP) that hybridizes to a conserved region of the 3 ' UTR, and the same arbitrary primers that were used in the first round. The products of this second round of amplifications were Southern blotted and hybridized with an oligonucleotide probe (Hb, indicated by the solid bar) that recognizes the LlHs 3' UTR. Dimorphic bands were extracted, cloned by the TA method (Invitrogen) , and sequenced. The arrows below indicate the relative positions and orientations of the 19 or 20 bp long flanking primers that were synthesized to match the non-Ll flanking DNA sequences (3FPa, 3FPb, and 5FP) . Figures 2A-2E. Identification of LID 1-6 by LI display and verification of their dimorphic status.

2A . LI display. The products of the second round of PCR amplifications were Southern blotted and probed with oligonucleotide Hb. Digital photographs (Kodak DC40) of the sections of the autoradiograms that depict LID 1-6 are shown. Each lane represents the results obtained from one individual. Ca-1, and 2, Caucasian 1 and 2; Ch, Chinese; Dr. Druze ; Py, Pygmy; Me, Melanesian. 2B . PCR amplification with primers ACA and 3FPa. For each

LID, 200 ng of genomic DNA were amplified with primers ACA and the LID-specific 3FPa. PCR products were separated electrophoretically; digital photographs and the ethidium bromide stained gels (Kodak DC40) are shown. We verified the ability of the 3FPa oligonucleotides to prime PCR in all individuals by amplifying genomic DNA with flanking primers 3FPa and 3FPb (Figure 1) . Bands of the expected size were evident in all individuals (not shown) confirming that the absence of bands in Figure 2B was not the result of the failure of a 3FPa to prime.

2C. Southern blot of Ace I digested genomic DNA hybridized with 3' flanking probes. The probes were generated by amplifying the non-LlHs 3 ' flanking DNA of the cloned LIDs with primers 3FPa and 3FPb, and tailing the products by the addition of [ -³²P]dCTP with terminal transferase. Digital photographs of the autoradiograms of the hybridized blots are depicted. Bands representing both the empty alleles (slower mobility) and the occupied alleles (faster mobility) can be seen in the blots hybridized with the 3' flanking probes from LID 1, 2, 4, 5. The two bands in the Ca-2 and Dr samples of the LID 1 blot are located very near to one another; their positions are indicated by lines. The absence of bands for the Ca-1 samples in the LID 2 and LID 4 blots was due to an insufficient loading of DNA.

2D. and 2E. PCR amplification of LID 1-6 with 5' and 3' flanking primers. The 5' flanking sequences of LID 1-4 and 6 were determined by amplifying the empty alleles from the Genome Walker kit (Clontech) using the LID-specific 3FPa primers. The 5 ' flanking sequence of LID 5 was obtained from Genbank (accession AC002122) . For each LID, 200 ng genomic DNA were amplified with the LID-specific primers 5FP and 3FPa. The arrowheads indicate the location of the amplified products of the empty alleles. The larger bands are the amplified products of the filled alleles. Digital photographs of the ethidium bromide stained gels, 2D. , or the autoradiograms of the gel after blotting and hybridization with probe Hb, 2E. , are shown.

Figure 3. Distribution of LID 1-6 among the 6 subjects. Presence or absence of LIDs were determined by LI display and either Southern blotting, PCR with one or two flanking primers, or both. The presence of a LID in a given individual is indicated by a + . LIDs were present in the heterozygous state in all cases.

Figures 4A and 4B. Quantification of LlHs subsets in the human genome . 4A . Southern blot quantification. Genomic DNA from i) individual Ca-2, ii) mouse LMTK^" cells, and iii) LMTK^" cells to which plasmid pLl .2A (which contains a Ta subst LlHs) was added were digested with Sau 3A I and Ace I to release the LlHs 3'

UTRs (4) . Samples ii) and iii) were mixed in varying ratios to represent 0, 700, 1050, 1400, and 2100 relative copies of LlHs per haploid genome . 1 μg samples of each were Southern blotted and hybridized to oligomer C, an LIHs-Ta specific probe (3) . The relative activity of the hybridized bands was measured on a phosphorimager (Molecular Dynamics) . Results indicate a relative copy number of 2250 for the Ca-2 band, and a linear relationship of copy number to signal in the standard lanes.

4B . BLAST search results. Genbank release 100.0 was searched with the MacVector 6.0 (Oxford Molecular Group) implementation of the BLAST program for similarity to the sequence of LRE-1 from bp 5914 to its end (4, 29) . Sequences that matched with a significance of p<e^"20 were selected. All sequences that had been identified because they had retrotransposed or because they had been specifically cloned as members of subset Ta were excluded on the basis of ascertainment bias. Ta elements represented 1.3% (7/556) of the selected sequences suggesting that 1,300 LlHs-Ta3 ' UTRs reside in the haploid human genome (0.013 x 100,000 total LlHs 3' UTRs/haploid genome) . Eight elements with the sequence ACG...A/G were also selected. The subset of elements, which we call Tb, includes element JH-28, a previously reported de novo LlHs insertion (30) . The presence of a G residue at position 6015 was previously shown to be characteristic of subset Ta elements (9) .

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

As set forth above, the present invention is directed to display techniques for identifying LINE-1 insertion site polymorphisms.

Our goals in developing LI displays were to design methods that 1) required minimal preparatory manipulation of the DNA samples, 2) resulted in the separation of individual insertion sites in a single step, and 3) would be adaptable to high throughput instrumentation. An exemplary method is outlined in Figure 1. We used the LIHs-Ta subset which includes actively transposing members and is defined by the presence of the sequence ACA in the 3' UTR at position 5930-5932 (nucleotide numbers refer to the element LRE-1 ) (4) . Elements with genomic consensus have GAG at this location (9).

We preformed LI display with 14 arbitrary primers on DNA isolated from 6 male individuals from diverse ethnic backgrounds. With each of the primers strongly hybridizing bands were evident on the autoradiograms. Some of the bands were present in the reactions of all 6 individuals while others were not (not shown) . We hypothesized that the variable bands represented dimorphic LIHs-Ta insertions sites which we called LINE Insertion Dimorphisms (LID) . Ten variable bands were cloned and sequenced for further analysis (Figure 2A shows 6 of the 10) . Each of the 10 clones was unique and consisted of the terminal 80 bp of a LlHs 3' UTR (64 bp of amplified sequence plus 16 bp from primer NP) , an A-rich region, and a region of 3' flanking sequence (not shown) . Only 3 nucleotide differences from the LRE-1 sequence were detected among the terminal 64 bp of LlHs 3' UTR sequence determined from each of the clones (2 were present in poly-A addition signals) . All 10 clones contained a 6015G suggesting that they were members of subset Ta (see below) . Next we amplified the genomic DNA of each individual with primers ACA and 3FPa. Each of the 3FPa primers was specific for the non-LlHs 3' flanking DNA of one of the cloned variable bands (Figure 1) and each of the amplifications was done with a single 3FPa primer. In 6 cases, the presence or absence of unique bands of the predicted size matched the pattern seen m LI display (Figures 2A and 2B) . In the other 4 cases, amplification of genomic DNA with primers ACA and 3FPa resulted in bands of identical length in all 6 individuals (not shown) . These 4 clones may represent monomorphic LlHs insertions .

To confirm the dimorphic status of LID 1-6, genomic DNA from each individual was digested with Ace I, blotted, and hybridized with non-LlHs 3' flanking probes. Chromosomes containing the LID occupied alleles are expected to display shorter bands than chromosomes containing empty alleles (Figure 1) . In four cases (LID 1, 2, 4 and 5) the pattern of bands was as expected (Figure 2C) . We were unable to confirm the dimorphic status of the other 2 insertion loci by Southern blotting. Hybridization by the LID 3 flanking probe revealed only a single weak band which was present in each individual, while hybridization with the LID 6 probe resulted m a smear

(Figure 2C) . We also obtained 5' flanking sequence from each of the empty insertion loci and amplified genomic DNA using the LID- specific flanking sequence primers 5FP and 3FPa (Figures 2D,

2E) . Amplification of empty alleles is expected to result in oands that are shorter than amplification of the occupied alleles by a size that depends on the length of the LlHs insertion (10) . In each case, both the empty alleles

(arrowheads) and the filled alleles were visible and the pattern of the bands was as expected. By DNA sequencing of the amplified bands we determined that LID 1-6 all contained the ACA sequence, indicating that they indeed are members of subset Ta. Figures 2C and 2D also indicate that the occupied allele was always present m the heterozygous state. Two of the LIDs (2 and 3) were present in only a single subject and each of the 6 subjects was unique on the basis of the presence or absence of

LID 1-6 (Figure 3) . These sites were also amplified from the genomes of one gorilla and one chimpanzee and found to be empty

(not shown) .

To estimate the number of LlHs dimorphisms present in an average human we first determined the total number of LIHs-Ta 3'

UTRs m the haploid genome. Quantitative Southern blotting revealed that 2250 were present (Figure A) . This result compares favorably to an estimate derived from a search of

Genbank (Figure 4B) and to the previous estimate, also based on quantitative Southern blotting, that 2% (80/4000) of full length elements belong to subset Ta (3) . We estimated the frequency of Ta element dimorphisms by counting the LI display bands obtained using the 14 arbitrary primers. An average of 10 variable, and

20 uniform bands were evident per individual (not shown) . Only

6 of the 10 cloned variable bands represent confirmed dimorphic loci, indicating that roughly 20% (0.6 x .0/30) of Ta sites in an individual are dimorphic, and 900 (2250 x 2 x 0.2) dimorphic loci exist n an average diploid human genome. The actual number of dimorphisms is greater than 900 because LI display cannot distinguish between individuals who are homozygous or heterozygous for occupied alleles (both types of individuals would show LI display bands) . We also preformed LI display on the same genomic samples with primer GAG (see Materials and Methods) instead of primer ACA, and 10 arbitrary primers. This revealed an average of 39 uniform but only 2 variable bands per individual (not shown) indicating that the rate of dimorphism of non-Ta, non-Tb (Figure A B) elements is significantly lower. We used LINE-1 display to isolate two LINE-1 tail length polymorphisms. One of these was bi-allelic and heterozygous in only 1 of 10 individuals while the second was multi-allelic and heterozygous in 2 of 6 individuals.

LIDs have great potential as markers for a variety of genomic studies. The insertion of a transposable element into a particular chromosomal locus likely represents a unique historic event (10) . Overwhelmingly, alleles bearing LlHs insertions are identical by descent, not just by state. This is not the case for most other types of genetic markers (11) . It is also reasonable to infer that the ancestral allele is the one without the insertion (12) . A possible role for LIHs-associated polymorphisms in the mapping of centromeres had been suggested

(13-14). The value of Alu dimorphisms has already been demonstrated (15-19) . Dimorphic Y family Alu insertions are estimated to be an average of 1.6 million years old (11) . Several findings suggest that many LlHs dimorphisms may have occurred more recently. More LlHs than Alu de novo insertions have been reported suggesting that the modern rate of LlHs expansion may exceed the rate for Alu ' s (3, 17) . Also, in the present small study, LID 1-6 were present in the heterozygous state m all individuals, two of six LIDs were present in only a single individual, and few LlHs 3' UTR nucleotide changes were detected. Thus Alu dimorphisms and LIDs may represent complementary sets of genomic markers. LlHs elements are not distributed randomly in the human genome (20-23) . LI display may help establish if the selection occurs at the time of transposition or thereafter. LI display will also be useful for investigating other questions of LlHs biology including the rate of somatic and germ-line transposition, and a possible role for LlHs m the development of cancer (24-25). Materials and Methods Genomic DNA. Melanesian (Me) , pygmy (Py) , Druze (Dr) , and Caucasian-1 (Ca-1) DNA were isolated from tissue culture cell lines GM10540, GM10492, GM11522, and GM05386 (Coriell Cell Repository) , respectively. Caucasian-2 (Ca-2) and Chinese (Ch) DNA were isolated from blood samples donated by the authors. All samples were derived from male individuals . Primate DNA was kindly provided by Dr. Anthony Furano. Genomic DNA preparation was preformed by a standard protocol (26) .

Oligonucleotides . Arbitrary oligonucleotide decamers were purchased from Operon (27) . The other oligonucleotides used in this study were prepared by the Center for Biologies Evaluation and Research core facility, or purchased from Life Technologies. The sequence of the LI display oligonucleotides were:

ACA 5 ' -CCTAATGCTAGATGACACA-3 ' NP 5 ' -GCACCAGCATGGCACA-3 '

Hb 5 ' -CCTGCACAATGTGCACATGTACCC-3 ' GAG 5 ' -CCTAATGCTAGATGACGAG-3 ' .

The sequence of the other oligonucleotides used in this study are available from the authors upon request .

Polymerase chain reaction. Three different types of PCR reactions were preformed:

1. LI display PCR. The first round of LI display PCR reactions were carried out with 25 ng genomic DNA, 0.5 μM primer ACA, and 0.3 μM decamer primer in 20 mM Tris pH 8.4 , 1.5 mM MgCl₂, 50 mM CaCl₂, 0.2 mM deoxynucleotides, and 2.5 U Taq polymerase for 40 cycles of 94°C for 30 sec, 36°C for 30 sec, and 72°C for 30 sec. The second round of reactions were carried out using the same conditions except that the primer NP was substituted for primer ACA, and the template consisted of 2.5 μl of the products of the first round reactions.

2. 3 ' flanking PCR. Amplifications with primer ACA and primer 3FPa (Figure 2B) were performed with 200 ng genomic DNA, 0.2 μM of each primer and an annealing temperature of 50°C. The rest of the conditions were the same as above. 3. 5' and 3' flanking PCR. To amplify the LID insertion sites using primers 5FP and 3FPa (Figures 2D and 2E) we utilized BIO-X-ACT DNA polymerase (GeneMate) , 200 ng genomic DNA, and 0.2 μM of each primer in OptiBuffer (GeneMate) . Reactions were carried out for 31-36 cycles of 59°C for 30 sec, and 68°C for between 3 and 6 minutes depending on the length of the expected products .

Cloning and sequencing of PCR products. LI display DNA fragments were isolated from agarose gels with the QIAquick gel extraction kit (Qiagen) and cloned by the TA method (Invitrogen) . DNA sequences were determined with either the SequiTherm EXCEL kit (Epicentre Technologies) using ^3:P-labeled primers, or with the Thermo Sequanase kit (Amersham) using ^JJP- labeled dideoxynucleotides . Sequence analyses and database searching were preformed with the MacVector program version 6.0 (Oxford Molecular Group) .

Southern blotting and Ta Ouantitation . PCR products were separated electrophoretically in a 3% 3:1 Nusieve agarose gel (FMC) and alkaline blotted onto a Nytran Plus membrane (Schleicher and Schuell) . Hybridizations were performed with ³²P-end-labeled oligonucleotides (10^q cpm/μg) in 5x SSPE/0.3% SDS/10 μg/ml salmon sperm DNA at 42°C overnight. The membranes were washed in 2x SSPE at 25°C for 15 minutes, 2x SSPE/0.1% SDS at 25° for 45 minutes, and twice in 0.5x SSPE/0.1% SDS at 42°C for 15 minutes. Southern analysis of genomic DNA was performed as described (28) . To quantify the number of Ta 3' UTRs in an average genome, 6 ng pLl .2A DNA was added to 4 μg of mouse LMTK^" DNA and digested with Sau 3AI and Ace I (4) . After digestion, the DNA was extracted with phenol/chloroform, ethanol precipitated, re-dissolved and the concentration determined again spectrophotometrically . The DNA was then diluted with a similarly prepared sample of mouse LMTK^" DNA (into which no plasmid DNA had been added) at relative copy numbers of 700, 1050, 1400, and 2100 copies of pLl .2A per haploid genome. 1 μg samples of each and 1 μg of similarly digested genomic DNA from individual Ca-2 were Southern blotted and hybridized to the LIHs-Ta specific oligonucleotide (oligomer C³) . Hybridization and washing conditions were as listed above.

The foregoing detailed description has been given for clearness of understanding only and no unnecessary limitations should be understood therefrom as modifications will be obvious to those skilled in the art.

REFERENCES

Each of the following citations is herein incorporated by reference:

1. Hwn, H.R., Roberts, J.W., Davidson, E.H. & Britten, R.J.

1986. Insertion and/or deletion of many repeated DNA sequences in human and higher ape evolution. Proc. Natl. Acad. Sci. U.S.A.

83:3875-3879. 2. Smit, A.F.A. 1996. The origin of interspersed repeats in the human genome. Curr. Opin. Genet. Dev. 6:743-748.

3. Sassa an, D.M. et al. 1997. Many human LI elements are capable of retrotransposition. Nature Genet. 16:37-43.

4. Dombroski, B.A., Mathias, S.L., Nanthakumar, E., Scott, A.F. & Kazazian Jr., H.H. 1991. Isolation of an active human transposable element. Science. 254:1805-1808.

5. Dombroski, B.A., Scott, A.F. & Kazazian Jr., H.H. 1993. Two additional potential retrotransposons isolated from a human LI subfamily that contains an active retrotransposable element. Proc. Natl. Acad. Sci. U.S.A. 90:6513-6517.

6. Holmes, S.E., Dombroski, B.A., Krebs, C, Boehm, C. & Kazazian Jr., H.H. 1994. A new retrotransposable human LI element from the LRE2 locus on chromosome lq produces a chimaeric insertion. Nature Genet. 7:143-148. 7. Woods-Samuels, P. et al. 1989. Characterization of a nondeleterious LI insertion in an intron of the human factor VIII gene and further evidence of open reading frames in functional LI elements. Genomics. 4:290-296. 8. Stoneking, M. 1997. The human genome project and molecular anthropology. Genome Res. 7:87-91. 9. Skowronski, J. , Fanning, T.G. & Singer, M.F. 1988. Unit- length line-1 transcripts in human teratocarcinoma cells. Mol. Cell. Biol. 8:1385-1397.

10. Batzer, M.A. & Deininger, P.L. 1991. A human-specific subfamily of Alu sequences. Genomics. 9:481-487.

11. Arcot, S.S. et al. 1996. Alu fossil relics-distribution and insertion polymorphism. Genome Res. 6:1084-1092.

12. Batzer, M.A. et al. 1994. African origin of human-specific polymorphic Alu insertions. Proc. Natl. Acad. Sci. USA. 91:12288-12292.

13. Prades, C, Laurent, A.M., Puechberty, J., Yurov, Y. & Roizes, G. 1996. SINE and LINE within human centromeres. J. Mol. Evol. 42:37-43.

14. Laurent, A.M., Marcais, B., Muleris, M. & Roizes, G. 1994. A rapid and simple method to isolate and characterize highly polymorphic markers from the centromeric regions of the human chromosomes. Nucl. Acids Res. 22:194-199.

15. Hammer, M., F. 1994. A recent insertion of an Alu element on the Y chromosome is a useful marker for human population studies. Mol. Biol. Evol. 11:749-761.

16. Zietkiewicz, E., Richer, C, Makalowski, W., Jurka, J. & Labuda, D. 1994. A young Alu subfamily amplified independently in human and African great apes lineages. Nucl. Acids Res. 22:5608-5612. 17. Tishkoff, S.A., Ruano, G., Kidd, J.R. & Kidd, K.K. 1996. Distribution and frequency of a polymorphic Alu insertion at the plasminogen activator locus in humans. Hum. Genet. 97:759-764.

18. Novick, G.E., et al. 1995. Polymorphic human specific Alu insertions as markers for human identification. Electrophoresis . 16:1596-1601.

19. Batzer, M.A. et al . 1996. Genetic variation of recent Alu insertions in human populations. J. Mol. Evol. 42:22-29.

20. Soriano, P., Meunier-Rotival, M. & Bernardi, G. 1983. The distribution of interspersed repeats in nonuniform and conserved in the mouse and human genomes. Proc. Natl. Acad. Sci. USA. 80:1816-1820. 21. Moyzis, R.K., et al. 1989. The distribution of interspersed repetitive DNA sequences in the human genome. Genomics . 4:273- 289.

22. Manuelidis, L. & Ward, D.C. 1984. Chromosomal and nuclear distribution of the Hindlll 1.9-kb human DNA repeat segment.

Chromosoma. 91:28-38.

23. Korenberg, J.R. & Rykowski, M.C. 1988. Human genome organization: Alu, Lines, and the molecular structure of metaphase chromosome bands. Cell. 53:391-400. 24. Brattauer, G.L., Cardiff, R.D. & Fanning, T.G. 1994. Expression of LINE-1 retrotransposons in human breast cancer. Cancer. 73:2333-2336.

25. Bratthauer, G.L. & Fanning, T.G. 1992. Active LINE-1 retrotransposons in human testicular cancer. Oncogene . 7:507- 510.

26. Sambrook, J. , Fritsch, E.F. & Maniatis, T. 1989. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.

27. Li, S.W., Nembhard, K.M., Prockop, D.J. & Khillan, J.S. 1996. Identification and cloning of integration site DNA by PCR.

BioTechniques . 20:356-358.

28. Church, G.M. & Gilbert, W. 1984. Genomic Sequencing. Proc. Natl. Acad. Sci. USA. 81:1991-1995.

29. Altschul, S.F., Gish, W., Miller, W. , Myers, E.W. & Lipman, D.J. 1990. Basic local alignment search tool. J. Mol. Biol.

215:403-410.

30. Kazazian, et al . 1988. Haemophilia A resulting from de novo insertion of LI sequences represents a novel mechanism for mutation in man. Nature (London). 332:164-166. 31. Swergold, G.D. 1990. Mol. Cell. Biol. 10:6718-6729.

32. Skowronski, J. , et al . 1988. Mol. Cell. Biol. 8:1385-1397.

33. Grimaldi, G., et al. 1984. EMBO J. 3:1753-1759.

34. Lewin, B. 1990. Retroviruses and Retroposons. In Genes IV. Oxford University Press, New York, N.Y. Chapter 34:672-690. 35. Fanning, T.G. Issued January 18, 1994. Antibodies to p40. U.S. Patent No. 5,280,108. 36. Charlieu, J.-P., Carter, D.A., Bellis, M. & Roizes, G. 1992. 3¹ Alu PCR: a simple and rapid method to isolate human polymorphic markers. Nucleic Acids Research. 20 ( 6) : 1333-1337.

Claims

WHAT IS CLAIMED IS:

1. A method for identifying LINE-1 (LI) insertion site dimorphisms in mammals comprising, isolating genomic DNA from two or more mammals of the same species, amplifying DNA of each DNA sample at an LI insertion site by PCR with a first primer that hybridizes specifically to a site in the Ll, and a second primer of arbitrary sequence that hybridizes to a sequence in non-Ll DNA flanking the Ll, and further amplifying the insertion site DNA obtained in the preceding step in a second PCR reaction with a nested primer that hybridizes specifically to a sequence in the amplified Ll DNA that is different from the site bound by said first primer, and with said second primer, and determining the relative sizes of the DNA products from said second PCR reaction, and identifying that the set of amplified Ll insertion site DNA products obtained from genomic DNA of one individual is different from that obtained from a different individual of the same species.

2. The method of claim 1 wherein the Ll insertion site dimorphisms are identified in DNA of two or more humans.

3. The method of claim 2 wherein the Ll is an LlHs of the Ta subset (L1HS-TA) .

4. The method of claim 3 wherein the 3' end of said first primer has the nucleotide sequence ACA.

5. The method of claim 3 wherein said first primer has the nucleotide sequence of the primer designated ACA.

6. The method of claim 1 wherein said second primer hybridizes to a sequence in non-Ll genomic DNA flanking the 3' end of an Ll.

7. The method of claim 1 wherein said nested primer hybridizes to a conserved sequence in Ll DNA.

8. The method of claim 1 wherein said nested primer has the nucleotide sequence of the primer designated NP.

9. The method of claim 1 wherein said the relative sizes of the DNA products from at least one of said PCR reactions are determined by gel electrophoresis and Southern blotting with a hybridization probe which hybridizes specifically to Ll DNA.

10. The method of claim 9 wherein said hybridization probe has the nucleotide sequence of the DNA molecule designated Hb.

11. The method of claim 1 wherein the differences between sets of amplified Ll insertion site DNA sequences obtained from genomic DNA of two or more mammals are due to differences in Ll insertion site locations or to differences in the lengths of Ll A-rich tails in the genomic DNA of said mammals.

12. The method of claim 11 wherein the differences between sets of amplified Ll insertion site DNA sequences from genomic

DNA of two or more mammals are due to differences in one or more Ll insertion site locations in the genomic DNA of said mammals.

13. The method of claim 12 wherein the mammals are humans, the Ll is LIHs-Ta, and the differences between amplified LlHs insertion site DNA sequences of two or more humans are due to differences in locations of one or more LIHs-Ta insertion sites in the genomic DNA of said humans.

14. The method of claim 1, further comprising, determining the nucleotide sequence of one or more of said DNA products of said second amplification reaction that are present in at least one individual but are absent in at least one other individual, and identifying a non-Ll nucleotide sequence that is present at a site flanking an Ll insertion site in the genomic DNA of at least one individual, subjecting the genomic DNA of two or more of said mammals of the same species to a third PCR amplification reaction with a first primer that hybridizes specifically to a site in the Ll DNA, and a second primer that hybridizes to said non-Ll sequence in genomic DNA flanking the Ll insertion site, and identifying a DNA product of said third PCR amplification that comprises an Ll DNA sequence, the identification of which is indicative of an Ll insertion site dimorphism in the genomic DNA of said mammals.

15. A method for forensic identification of an individual, body tissue, body fluid, or body part, comprising isolating genomic DNA from said individual, tissue, body fluid, or body part, and using the method of claim 1 to detect one or more Ll insertion site dimorphisms in said DNA which identify said individual, tissues, body fluid, or body part.

16. A method for identifying the donor of donated cells or organs, comprising isolating genomic DNA from said cells or organs and from suspected donors, and using the method of claim 1 to detect one or more Ll insertion site dimorphisms in said DNA from cells or organs which match those of the donor so as to identify the donor of said cells or organs.

17. A method for identifying the ethnic or genealogical background of an individual comprising isolating genomic DNA from said individual and from other individuals of various ethnic or genealogical backgrounds, and using the method of claim 1 to detect one or more Ll insertion site dimorphisms in the DNA from said individual which match those present in said individuals of known ethnic or genealogical backgrounds so as to identify the ethnic or genealogical background of said individual .

18. A method for identifying an individual with genetic predisposition to be susceptible to a disease, comprising isolating genomic DNA from said individual, and using the method of claim 1 to detect one or more Ll insertion site dimorphisms in said DNA which match those characteristically present in DNA of individuals who are susceptible to the disease so as to identify said individual as one who is also susceptible to the disease .

19. A method for identifying an individual with genetic predisposition to respond to a medical treatment, comprising isolating genomic DNA from said individual, and using the method of claim 1 to detect one or more Ll insertion site dimorphisms in said DNA which match those characteristically present in DNA of individuals who are genetically predisposed to respond to the medical treatment so as to identify said individual as one who is also genetically predisposed to respond to said medical treatment .

20. A kit for identifying Ll insertion site dimorphisms in mammals, comprising a first PCR primer that hybridizes specifically to a site in the Ll, one or more primers of arbitrary sequence that hybridize to a sequence in non-Ll DNA flanking the Ll, and a third, nested primer that hybridizes specifically to a sequence in the amplified Ll DNA that is different from the site bound by said first primer.